Human iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) dioxygenases hydroxylate unactivated aliphatic carbon centers in reactions that are fundamentally important to central life processes (e.g., metabolism and its regulation, transcription, epigenetic inheritance) and relevant to several diseases. Plant, fungi, and bacteria have diversified the Fe/2OG-oxygenase platform for a bewildering array of oxidative transformations that include halogenations, cyclizations, dehydrogenations and stereoinversions of aliphatic carbon centers. As the biosynthetic machinery generating a large number of important natural-product drugs are replete with such Fe/2OG oxygenases, the ability to reprogram them by either rational or directed-evolution approaches would enable microbial/enzy- matic production of novel drug compounds. In this project, we will use in vivo and in vitro selection methods to engineer outcome-altered variants of Fe/2OG oxygenases on pathways to antibiotic and anesthetic drugs. Our extensively validated kinetic and spectroscopic approaches to structural and functional analysis of these enzymes will be applied in combination with two new, innovative structural methods to rationalize the reprogramming in precise structural and mechanistic terms. The project will thus provide both enzymes for production of new potential drugs and mechanistic insight to enable more rational reprogramming of these and other Fe/2OG oxygenases.